ABSTRACT Campylobacter jejuni is a primary bacterial cause of gastroenteritis in the United States and throughout the world, with 140 million cases worldwide and 1.3 million cases of C. jejuni gastroenteritis in the U.S. each year. This infection leads to >30,000 deaths annually, primarily in children <5 years old in underdeveloped countries. Most cases of C. jejuni disease are sporadic and result from contaminated food (especially poultry) and exposure to environmental waters. Some C. jejuni infections (~1/1,000) lead to the development of Guillain- Barr Syndrome, the leading cause of acute paralysis in the world. Despite the high prevalence of Campylobacter disease and nearly 40 years of research, the mechanisms by which C. jejuni causes disease remain incompletely understood and severely understudied. Numerous bacteria, including human pathogens, use the second messenger cyclic-di-GMP (c-di-GMP) to regulate metabolic and virulence-related characteristics. Typically, c-di-GMP is synthesized by diguanylate cyclases (DGCs) containing GGDEF domains, and degraded by phosphodiesterases (PDEs) with EAL or HD- GYP domains. Accumulated c-di-GMP binds to diverse receptors that mediate the downstream regulatory effects of c-di-GMP. Bacterial c-di-GMP signaling networks can be quite complex; some bacteria have dozens of DGCs and PDEs that are thought to integrate numerous upstream signals and adjust protein expression or activity accordingly. In contrast, the predicted C. jejuni c-di-GMP network consists of a single DGC, the bile- resistance response regulator CbrR. This is the first study of c-di-GMP signaling in C. jejuni. Overall Hypothesis: C. jejuni uses c-di-GMP signaling to modulate bile resistance, motility/chemotaxis, and biofilm formation, by means of the divergent diguanylate cyclase response regulator CbrR. We propose a detailed study of c-di-GMP-mediated gene regulation in C. jejuni, focusing on the mechanism by which the diguanylate cyclase CbrR controls the expression of bile resistance, motility/chemotaxis, and biofilm formation. We will use a combination of genetic, biochemical, and RNA-Seq approaches to achieve the goals outlined in these two specific aims: Aim 1) Define the role of CbrR-produced c-di-GMP in C. jejuni bile resistance, motility/chemotaxis, and biofilm formation; and Aim 2) Determine the genes that are regulated by CbrR. Together, these will lay the groundwork for our long-term goal of interrupting this novel signaling network and interrupting inter-host transmission and pathogenesis in humans.